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the chance to identify and directly examine what happens within the differ-
entiated nucleus as reprogramming begins (
Pereira et al., 2008
). As fusion is
possible between cells of different species (
Harris, 1965; Harris & Watkins,
1965; Harris, Watkins, Campbell, Evans, & Ford, 1965
), it is possible to
monitor changes in gene expression using species-specific primers so as to
discriminate the events that occur in the reprogrammer (ESC) and the
reprogrammed (differentiated) targets (
Pereira & Fisher, 2009
). Such track-
ing is not possible with cells that belong to the same species, so a range of
genetically engineered target cells have become available that express
markers (such as OCT4-GFP) in response to pluripotent conversion (e.g.,
Han et al., 2008
). Using such tools to generate heterokaryons with ESCs, it
has been possible to chart the earliest events that occur
en route
to successful
reprogramming (e.g.,
Bhutani et al., 2010; Do & Sch¨ ler, 2004; Han et al.,
2008; Tada et al., 2001
) and to show that reprogramming is initiated at a
stage immediately prior to nuclear fusion and cell division.
Reprogramming can also be induced by the forced expression of a com-
bination of pluripotency-associated factors. This was first shown by
Takahashi and Yamanaka (2006)
who reported that the expression of four
transcription factors were sufficient to generate ESC-like pluripotent cells
from differentiated targets. Since this time, Yamanaka's strategy has been
modified and extended so that factor-based induction can be used to repro-
gram targets to become specific somatic stem cell types (reviewed in
Pereira,
Lemischka, & Moore, 2012
). Despite improvements in generating iPSCs,
the efficiency of successful conversion using this approach remains generally
low (0.0001-2%). Reprogramming using iPS also takes several days to
several weeks to accomplish. As discussed in
Section 4
, conversion in the
iPS system is thought to depend at least partially on yet-to-be-uncovered
stochastic events that may facilitate epigenetic reprogramming. These events
might be present in only a subset of targets or could occur in most cells pro-
vided the inducers are allowed sufficient time and opportunity for cell
division (
Hanna et al., 2009
). As an excess of reprogramming factors is
known to be able to trigger cell-cycle arrest, death or inappropriate differ-
entiation in transfected cells, achieving the right balance of reprogramming
factors appears to be critical (
Banito et al., 2009; Kawamura et al., 2009;
Li et al., 2009
). Different forms of genomic impairment also seem to block
successful reprogramming, as somatic cells with different levels of DNA
damage are eliminated by p53-dependent pathway (
Mari´n et al., 2009
).
Elimination of p53 allows more efficient reprogramming; however, the
resulting iPSCs
show greater
levels of chromosomal
instability. As
